The intestinal epithelium is one of the fastest regenerative tissues in the body. Regeneration is sustained by a group of stem cells, termed crypt base columnar cells (CBCs), at the bottom of the intestinal crypt. In the past 5 years, studies of CBCs have helped transform the dogma of stem cell biology. Rather than being largely quiescent, undergoing asymmetric division, and following the unidirectional differentiation hierarchy, CBCs are highly proliferative, capable of symmetric division, and replaceable by more differentiated cell types. Since then, similar stem cell populations and regulatory principles have been identified in many other tissues. However, all these discoveries converge on a central question: how does the stem cell niche control plasticity in order to keep a constant number of CBCs? Since proliferative CBCs have been linked to colon cancer, and stem cell transplants are being used clinically for treating a variety of diseases, it is importantto understand the underlying control. Cellular dynamics Innovative multiphoton imaging and laser ablation technologies will be used to investigate how cells divide, move and recover loss inside the stem cell niche, focusing on four fundamental questions: (1) are CBCs capable of both symmetric and asymmetric division, (2) do individual CBCs in the same niche have equal proliferative potential, (3) how does the niche recover from cell loss, and (4) how do cells outside the niche dedifferentiate and reenter the niche? Signaling dynamics Using the 3D intestinal organoid assay, a systematic study will be carried out to search for feedback and crosstalk mechanisms among major signaling pathways. Dynamical systems analysis will be performed to understand their impact on the niche control circuitry. Integration and Validation To integrate experimental findings and computational insights, a stochastic, multiscale model will be built that captures cellular division, movement and signaling events in the stem cell niche. This model will be used to test hypotheses on the niche control circuitry. A novel engraftment assay through injection of genetically engineered stem cells into blastocysts has been developed for in vivo validation. Innovation Novel in vivo GI imaging techniques have been developed by using openable abdominal window, 3-D printed intestinal support, labeled vasculature roadmap, and tracking algorithms. Through the window, a special laser can ablate a single cell inside the niche without damaging the surrounding tissue. A computational framework will integrate dynamical systems analysis and multiscale modeling to study the regulatory circuitry that controls cellular and signaling dynamics inside the niche. Predictions will be validated in novel chimeric mice with intestinal crypts derived from blastocyst-injected cells. Preliminary findings The stem cell niche undergoes extensive reorganization after loss of one CBC, attesting to its dynamic nature. Various feedback and crosstalk mechanisms have been identified to improve robustness.